This invention relates to a novel procedure for solubilizing amphiphilic drugs. In another aspect, this invention relates to improved methods of Amphotericin B liposome preparation. In another aspect this invention relates to improved methods of producing liposomes by a commercially feasible process. This invention also relates to liposomal Amphotericin B having reduced toxicity and to a new method of treatment with liposomal Amphotericin B.
Systemic fungal infections are a major cause of mortality in cancer patients and other immunocompromised individuals. Unfortunately, fungal infections very often defy treatment because the few drugs that destroy fungi are extremely toxic to the host. Because of the drugs"" toxicity, the lowest possible effective doses should be given. Unfortunately, because the drugs are diluted in the blood, and because large amounts of the drugs are degraded, or excreted or taken up by uninfected tissue, large doses actually are and must be given if the treatment is to be effective.
The preferred treatment for systemic fungal infections is primarily limited to two groups of drugs: the polyene antibiotics such as Amphotericin B and nystatin, and the imidazoles, such as ketaconazole and miconazole. The polyene antifungal antibiotics readily bind to sterol components of host cells causing disruption of the membrane, cell permeability and lysis. Amphotericin B has thus been associated with acute hemolytic crisis. Further, because it is particularly toxic to kidney tissue, it has been associated with irreversible renal damage and even kidney failure, at therapeutic dosage levels. Medoff, G., Kabayashi, G. (1980) Strategies in treatment of systemic fungal infections. New England Journal of Medicine 302: 145-55; Cohen, J. (1982) Antifungal chemotherapy. Lancet ii: 532-37; Graybill, J. R., Craven, P. C. (1983) Antifungal agents used in systemic mycosis: activity and therapeutic use. Drugs 25: 41-62.
It is a major goal of medical research to overcome the problems presented by the need for compromise between dosages high enough to control infection on the one hand, and unacceptable damage to healthy tissues on the other. Recently it has been discovered that needed doses of medicine can be delivered to diseased tissue while bypassing healthy tissue using certain liposomal formulations. Additionally, it has been recognized that medication can be incorporated into liposomes, microscopic delivery vesicles made, in part, of phospholipids. See U.S. Pat. No. 4,663,167xe2x80x94Composition and Method for Treatment of Disseminated Fungal Infections in Mammals, incorporated here by reference, and Vestar application Ser. No. 899,064 entitled xe2x80x9cImproved Treatment of Systemic Fungal Infections with Phospholipid Particles Encapsulating Polyene Antifungal Antibioticsxe2x80x9d (now U.S. Pat. No. 5,043,107), also incorporated by reference, . . . xe2x80x9dwhich discloses liposomal delivery vesicles made, in part, from phospholipids.
Phospholipids form closed, fluid filled spheres when mixed with water. Phospholipid molecules are polar, having a hydrophilic ionizable head, and a hydrophobic tail consisting of long fatty acid chains. Thus, when sufficient phospholipid molecules are present with water, the tails spontaneously herd together to exclude the water while the hydrophilic phosphate heads form bonds with the water.
The result is a bilayer in which the fatty acid tails point into the newly formed membrane""s interior and the polar heads point toward the aqueous medium. The polar heads at one surface of the membrane point toward the liposome""s aqueous interior and those at the other surface point toward the aqueous exterior environment. It is this chemical tendency to form liquid filled spheres that allows the liposome to be loaded with medication. As the liposomes form, water soluble molecules will be incorporated into the aqueous interior, and lipophilic molecules will tend to be incorporated into the lipid bilayer. Liposomes may be either multilamellar, like an onion with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding an entirely liquid center.
In studies of mice, Amphotericin B incorporated into liposomes has been shown to treat systemic fungal infections more effectively than when given as the free drug. Liposomes are not themselves toxic, and they protect their loads from being degraded or diluted. Thus, liposomes are thought to deliver concentrated doses of antifungal antibiotic at the diseased tissue without the toxicity that would otherwise be associated with freely circulating drug. Therefore, liposomal Amphotericin B drug doses can exceed the maximum tolerated dose of free Amphotericin B. Mehta, R. (1982), Liposomal aphotericin B is toxic to fungal cells but not to mammalian cells. Biochimica et Biophysica Acta 770: 230-34. Liposomal encapsulated Amphotericin B has also been shown to be an effective treatment for murine systemic fungal infections, including Candidiasis, Cryptococcosis, and Histoplasmosis. Graybill, J. R. et al (1983) Treatment of murine cryptococcosis with liposomal associated Amphotericin B. Journal of Infectious Diseases 145: 748-52; Taylor, R. L. et al (1982) Amphotericin B in liposomes: A novel therapy for histoplasmosis. American Review of Respiratory Diseases 125: 610-611.
Liposomal Amphotericin B has also shown effectiveness in human patients, life saving when other treatments have failed, including freely circulating Amphotericin B. Systemic fungal infections are seen most commonly in people whose immune systems are compromised by disease or immunosuppressive drug therapy. As previously mentioned, these infections are a common cause of death to victims of acquired immune deficiency syndrome and to cancer patients undergoing chemotherapy. The causative agents of these fungal infections are often endogenous fungi that would be rendered harmless but for the patient""s impaired resistance. Lopez-Berestein, G. et al (1987) Treatment of hepatosplenic Candidiasis with liposomal Amphotericin B. Journal of Clinical Oncology 5:310-17.
Unfortunately, because of the chemical properties of the polyene antifungal antibiotics, it has heretofore not been possible to produce liposomal Amphotericin B in commercial quantities. These antifungal agents are called polyenes because they contain three to seven conjugated double bonds in the aliphatic chain making up a large lactone ring. The double bonds are incorporated into one side of the ring of 26 to 44 carbon atoms and along the opposite side of the ring 6 to 12 hydroxyl groups are present. Additionally, these molecules contain specific carboxylic acid groups and amine groups. Amphotericin B and nystatin, for example, possess both an aminosugar and a carboxylic acid group. The polyene regions of the molecules are of course hydrophobic and lipophilic while the polyol and ionizable regions are hydrophilic and lipophobic. As such, these molecules are called amphiphilic. Additionally, because of the carboxylic group and the amine group, Amphotericin B can act as a Lowry-Brxcfx86nsted acid or proton donor, or as a Lowry-Brxcfx86nsted base or proton acceptor. The combination of these functionalities makes polyenes very poorly soluble in water and most organic solvents. Bennett, J. E. (1974) Chemotherapy of systemic mycoses. New England J. Medicine 290: 320-23.
It has been the persistent problem of insolubility of the polyene antifungal antibiotics in general and of Amphotericin B in particular that has previously limited the prior art. Liposomal Amphotericin B formation was heretofore limited to generally two methods, described below, neither feasible for commercial scale production and neither showing long-term stability or as great a reduction in toxicity as the preparations herein described.
One prior method requires that the Amphotericin B be first dissolved in large volumes of volatile organic solvent such as methanol. To that solution would then be added the lipid mixture dissolved in a volatile organic solvent such as methanol and/or chloroform. The solvents would then have to be removed from the mixture to form a lipid-Amphotericin B film. Removal of solvents could be accomplished by a variety of methods but usually by evaporation to dryness in a round bottom flask under vacuum or nitrogen. Taylor, R. L. (1980) Amphotericin B in liposomes: A novel therapy for histoplasmosis. Am. Review Respiratory Disease 125: 610-11; Graybill, J. R. et al (1982) Treatment of murine crytococcosis diseases. J. Infectious Diseases 145: 748-52; Lopez-Berestein, G. (1983) Treatment and prophylaxis in disseminated infection due to Candida albicans in mice with liposome-encapsulated Amphotericin B. J. Infectious Diseases 147: 939-45; U.S. Pat. No. 4,663,167xe2x80x94Composition and method for treatment of disseminated fungal infections in mammals. The prior art methods thus required removal of large volumes of organic solvent, and preparation of a lipid-Amphotericin B film, an extra step eliminated by the present invention. Moreover, the prior art methods were thus practicable only in discrete batches and were not amenable to the continuous flow process of the current invention. These are two disadvantages which the industry has long attempted to overcome.
In another method of forming liposomal Amphotericin B, the lipid mixture is dissolved in chloroform or another solvent and deposited and dried on the sides of a round bottom flask or vesicle surface. A solution of Amphotericin B dissolved in a small amount of dimethyl sulfoxide would then be added to the previously deposited lipid film. The resulting preparation would thereafter have to be dialyzed against a buffered saline or other solution to remove the dimethyl sulfoxide and non-intercalated Amphotericin B. The procedure was extremely time consuming and expensive, and typically resulted in incorporation of only 70% of the initial Amphotericin B. Tremblay, C. et al (1984) Efficacy of liposome-intercalated Amphotericin B in treatment of systemic Candidiasis in mice. Antimicrobial Agents and Chemotherapy 26: 170-73.
Prior to the present invention, it was not possible to dissolve Amphotericin B in small quantities of volatile solvent, such that scaled-up production could be commercially feasible. The present invention also enables the dissolved Amphotericin Bxe2x80x94phospholipid liposomal solution to be spray dried, thus making commercial quantities practical by elimination of the elaborate and time consuming steps detailed above.
Accordingly, one object of the present invention is to provide an improved process for the solubilization of amphophilic drugs.
Another object of the present invention is to provide an improved process for the encapsulation of polyene antifungal antibiotics into liposomes.
More specifically, an object of the present invention is to provide an improved process for the formation of liposomal Amphotericin B.
Yet another object of the present invention is to provide a commercially feasible process for the production of liposomal Amphotericin B.
Yet another object of the present invention is to provide a process for the formation of liposomal Amphotericin B with reduced toxicity.
A further object of the present invention is to provide new methods of treatment with Amphotericin B.
The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.
According to the present invention, the steps of either evaporation of the large volumes of volatile solvent into which the antibiotic is at least minimally soluble, or of removing a nonvolatile solvent in which the antibiotic and lipid has been dissolved, typically by dialysis, have been eliminated. Instead, the present invention provides a new and useful lipophilic charge complex of Amphotericin B which overcomes the previous problems of insolubility. Additionally, the present invention provides a new and useful process for the production of Amphotericin B liposomes suitable for scaling up production to commercial quantities. Additionally, the invention provides a method for production of Amphotericin B liposomes of increased stability and decreased toxicity.
In one preferred embodiment of this invention, the new soluble complex is formed between Amphotericin B and distearoyl-phosphatidylglycerol which has been protonated during dissolution in a solution of chloroform and methanol acidified to a pH of about 1.0 to 3.0. The Amphotericin Bxe2x80x94phospholipid complex, while in solution in the small amount of acidified chloroform and methanol, can be mixed with phosphatidylcholine and cholesterol and reproducibly spray dried under controlled conditions to yield a lipid powder which is readily processed into liposomes, using an aqueous buffer solution having a pH such that the pH of the final solution is below about 5.5, preferably between about 4.5 and 5.5. Accordingly, the present invention allows commercial scaling up of new materials for liposomal production. Further, the liposomes formed with this invention can be lyophilized and stored for later rehydration and injection without significant change in size or toxicity. The advantages of the present invention, will become clear after considering the following detailed description.
Distearoylphosphatidylglycerol, or other homologous phosphatidylglycerols, such as dilaurylphosphatidylglycerol, dimyristoylphosphatidylglycerol or others, is dissolved in an equal volume solution of chloroform and methanol and acidified before forming the described soluble complex with Amphotericin B. Thus, 2.5 N hydrochloric acid is added to the distearoylphosphatidylglycerol sodium salt solution in methanol:chloroform (1:1) to adjust the pH to between 1.0 and 3.0, as measured on prewetted pH paper, prior to mixing with Amphotericin B, and thereby acidifying the phospholipid. The Amphotericin B, or other polyene such as a tetraene, pentaene, or hexaene, is suspended in an equivolume solution of chloroform and methanol and then added to the acidic distearoylphosphatidylglycerol solution. Complex formation is facilitated by briefly warming the solution to about 65xc2x0 Centigrade. At this stage, the concentration of Amphotericin B may be in excess of 45 mg/ml.
In the resulting solution additional lipids such as phosphatidylcholines may also be dissolved. Cholesterol or another sterol, such as ergosterol, stigmosterol, or androsterone, is included to improve the stability of the resulting liposomes, and thus maintain the liposome intact during circulation in the bloodstream. The solution is a translucent orange. Typically, the Amphotericin B concentration in the final solution is greater than 25 mg/ml, and the total dissolved solid material 15-20 percent by weight.
Among the additional phosphatidylcholines that may be dissolved in the Amphotericin B, acidic phosphatidylglycerol complex solution, hydrogenated egg phosphatidycholine, hydrogenated soya lecithin and distearoyl or dipalmitoyl phosphatidylcholine are preferred such materials. Hydrogenated natural phospholipids or saturated aliphatic phospholipids are believed to work well because the lack of double bonds in the side chains is thought to render the liposomes resistant to oxidation and more physically stable.
The organic solvents may be removed from the solution by rotary evaporation in a round bottom flask leaving a dry film comprised of the complex and other lipid materials. Other equivalent methods of solvent removal are also suitable, such as drying under vacuum. Alternately, the solution can be applied to a spray dryer and solvent removed in a continuous process to produce large quantities of a free flowing yellow powder for liposome preparation. This novel complex thus provides the long awaited continuous production capability sought by industry.
After the last traces of organic solvent have been removed, the dried lipid complex powder may be stored for later use as a starting material for liposome preparation. This product affords the stability necessary for storage previously unavailable to the industry. Thus, the initial chemical steps need not be undertaken each time liposomes are desired.
Liposome preparation is accomplished by first hydrating appropriate quantities of the lipid complex powder with an aqueous buffer, preferably at about 65xc2x0 C. Aqueous buffer solutions may also contain salts such as sodium chloride, or saccharides such as dextrose or lactose, to achieve any desired osmolarity. The pH of the solution is carefully controlled to achieve a final solution having a pH of about 5.5 or less, generally between about 2.0 and 5.5, preferably between about 4.5 and 5.5.
Liposomes are then formed by the application of shearing force. Typically shearing force can be applied using sonification or homogenization, or by freezing and thawing, dialyzing away a detergent solution from lipids, or other known methods used to prepare liposomes. The size of the liposomes, as well as whether they are multilamellar or unilamellar, can be controlled using a variety of known techniques including the duration of sonication. See Gregoriadis, G. A. Simple Procedure to Preparing Liposomes Capable of High Encapsulation Efficiency Under Mild Conditions. Liposome Technology, Gregoriadis, G. A. (ed.) CRC Press: Boca Raton, Florida (1983). The present invention is adaptable, in particular, to scaling up production of the small unilamellar liposomes disclosed in pending Vestar, Inc. application Ser. No. 899,064xe2x80x94Improved Treatment of Systemic Fungal Infections with Phospholipid Particles Encapsulating Polyene Antifungal Antibiotics, incorporated here by reference. Such small liposomes can be sterilized by filtration since their diameter is less than 0.2 xcexcm. Virtually all of the initial Amphotericin B becomes associated with the liposome fraction when this technique is employed.
These liposome preparations, when formed in saccharide solutions such as 9% lactose, may be lyophilized in vials under suitable conditions to form a dried yellow cake or plug of material. At a later time water may be introduced into the vial to redissolve the solid cake and form a suspension of Amphotericin B liposomes suitable for injection. Lyophilization thus affords the clinician the significant advantage of increased convenience.
Amphotericin B, and other polyene antifungal antibiotics, are amphiphilic. One side of the macrocyclic compound is composed of a series of unsubstituted hydrocarbons with double bonds while the opposing side is substituted with hydroxyl groups. Thus, the molecules tend to exhibit polarity, one side lipophilic and hydrophobic, the other side lipophobic and hydrophilic.
Furthermore, Amphotericin B has one carboxyl group, a Lowry-Brxcfx86nsted acid, and one amine group, a Lowry-Brxcfx86nsted base. Therefore, in a neutral pH range of from 5 to 9 the carboxyl group tends to give up a proton while the amine group tends to accept that proton. The net result is that the molecule remains neutral and uncharged while at the same time having two ionized or charged groupsxe2x80x94a negatively charged carboxyl group and a positively charged amine group. In that same neutral pH range phospholipid molecules, such as distearoylphosphatidylglycerol are charged. They have an ionized phosphate group giving the molecule a negative charge. Equally, such phospholipid molecules are amphiphilic in that the long alphatic tails are hydrophobic and lipophilic while the ionizable phosphate head is, of course, hydrophilic and lipophobic.
When, however, the phospholipid molecule is solubilized in a protic solvent with a pH between about 1.0 and 3.0, the phospholipid molecule tends to accept a proton and thus form a comparatively neutral molecule. When Amphotericin B is then added to the above acidified solution, the proton of the phosphate group will tend to be transferred to the carboxyl group of the Amphotericin B. The result is that the Amphotericin B molecule will have a net positive charge. Concomitantly, the phospholipid""s phosphate group will give up a proton and become negatively charged. The thus formed, oppositely charged, molecules attract; their oppositely charged groups forming an ion pair.
The molecular attraction between Amphotericin B and phosphatidylglycerol is thus greatly increased. The aliphatic hydrocarbon chains of the phospholipids are attracted by hydrophobic interactions to the long chain of unsubstituted double bonds of the polyene. In the specific instance of Amphotericin B, the molecule is a heptaene with seven double bonded carbons along an unsubstituted section of 16 carbon atoms. In the specific instance of distearoylphosphatidylglycerol there are 16 unsubstituted methylene groups between the ester group and terminal methyl group.
In addition to the hydrophobic interaction, the ionized groups form a strong association. In the protonating environment above described, the Amphotericin B will have a positive charge and the phosphatidylglycerol will have a negative charge. Thus, the phosphatidylglycerol and the Amphotericin B will form a strong association.
The strongly associated complex so formed is highly soluble in small amounts of organic solvent. Thus, the disadvantages of the aforementioned prior art methods have been overcome. See, for example, U.S. Pat. No. 4,663,167, May 5, 1987, Lopez-Berestein et al. Accordingly, the present invention provides a procedure for scaling-up production which the industry has long awaited.
A further aspect of the present invention is that the Amphotericin Bxe2x80x94phosphatidylglycerol complex will associate with phosphatidylcholine and cholesterol during formulation and will not precipitate out of the organic solvent solution. Furthermore, if the pH of this organic solvent is maintained at 4.5 or less, the complex continues to remain stable and strongly associated. During liposome hydration, the pH of the aqueous buffer is controlled to give a solution having a final pH preferably between about 4.5 and 5.5. At that pH, the Amphotericin B-lipid complex is highly stable and has a high affinity for the lipid bilayer into which it becomes inserted. The result is a decrease in acute toxicity as demonstrated by the following Chart 1:
Thus, a significant decrease in toxicity is attained using the present invention. The decrease in toxicity allows for an increase in the therapeutic dosages that may be safely administered and thus offers greatly improved methods for Amphotericin B treatment. See U.S. Pat. No. 4,663,167, May 5, 1987, Lopez-Berestein et al.
Furthermore, the associated complex is highly stable during storage. The liposomes formed from either a film, or spray dried powder, after hydration with a saccharide buffer, may be lyophilized. The lyophilized cake can be stored preferably in a sterile lyophilization vial and later rehydrated with sterile water for injection. The reconstituted liposomes retain therapeutic efficacy.